1. Field of Invention
This invention relates to orifices used to electrically sense the volume of particles in a fluid suspension flowing through the orifice.
2. Description of Prior Art
This background is provided to clarify the problems solved and the improvements provided by my invention.
In U.S. Pat. No. 2,656,508 issued in 1953 Coulter described his invention for a xe2x80x9cMeans for counting particles suspended in a fluidxe2x80x9d which provided an entirely new method of blood cell counting and sizing that was automatic, accurate, and reliable. Coulter""s idea was to pass-particles suspended in a conducting electrolyte through a small diameter orifice of short length. The electrical resistence of the particle must be greater than that of the electrolyte. A constant current was maintained across the orifice by two electrodes, one on each side of it. Then, as each particle traversed the orifice it displaced electrolyte, thereby producing an increase in resistance. This resistance pulse is observed as a voltage pulse. These voltage pulses are measured across the two electrodes, amplified, and those greater than a minimum or threshold value are counted with electronic scalers. FIG. 1 shows this basic resistance pulse device according to Coulter.
Coulter stated that the pulses are directly proportional to the volume of the particles, and thus the measured pulse height distributions should directly correspond to the volume distributions of the measured particles. However, this did not prove to be true. An excellent review of investigations into the causes for this discrepance and ways to eliminate or reduce it is presented by Volker Kachel in Chapter 4 of Flow Cytometry and Sorting, 2nd Edition, Wiley-Liss, 1990.
Two sources for the variable particle volume sensitivity have been identified. One is the non-uniform electrical field present within and adjacent to the orifice which produces electrical pulses of different heights and different time durations for particles of the same shape and volume which travel along different paths through the orifice. This effect is especially pronounced for particles traveling near entry and exit corners of the orifice where the electric field is concentrated and more intense.
A second source of volume signal error is produced by particles which have already passed through the orifice. These are present in the recirculating fluid at the downstream side of the orifice caused by the orifice effluent jet flow. When these particles pass near the exit corners of the orifice they produce pulses which are smaller in magnitude and longer in time duration than those produced by particles passing through the orifice.
The artifacts due to the non-uniform particle volume sensitivity of an orifice and the pulses produced by various particle paths through the orifice as well as those pulses produced by particles recirculating downstream of the orifice are shown on FIGS. 2(a), (aa), and (ab) respectively taken from the referenced work by Volker Kachel. It is clear that these artifacts can grossly distort the true particle volume distribution curves and true particle counts.
Various means to reduce or eliminate these artifacts have been devised. Spielman in J. Colloid Interface Sci., 26:175-182, 1968 described use of hydrodynamic focusing to virtually eliminate the effects of non-uniform sensitivity within the orifice by use of a sheath flow of particle-free electrolyte to carry the sample suspension through the center of the orifice. This sheath flow rate is in the order of 100 times that of the sample suspension flow rate. Thus the diameter of the sample stream is about 10% of the orifice diameter. This results in all the particles passing through a region of highly uniform particle volume sensitivity. This resulted in the improvement in erythrocyte volume sizing accuracy shown in FIG. 3 taken from Thom and Kachel in Blut 21:48-50, 1970.
The artifacts due to particles recirculating immediately downstream of the orifice are totally eliminated by capturing the effluent jet in a catcher tube and flushing the exit zone of the orifice with a particle-free electrolyte which also exits via the catcher tube. This rear sheath flow eliminates the recirculating particles which are especially troublesome when sizing and counting both small and large particles simultaneously. An instrument for counting both platelets and erythrocytes which utilizes both front and rear sheath flows was described by Haynes in Blood Cells, 6: 201-213, 1980. FIG. 4 shows the fluidic design of another instrument utilizing front and rear sheath flows.
FIG. 5 shows potential sensing electrodes located within the orifice itself in a zone having relatively uniform sensitivity and remote from recirculating particles. Leif and Thomas in Clin. Chem., 19:853-870, 1973 as well as Salzman et al in Biophys. Soc. Abstr., 17:3029, 1973 describe these orifices and their performance. This idea virtually eliminate particle volume centering artifacts and totally eliminates the recirculating particle artifact.
Karuhn et al in Powder Technol., 11:157-171, 1975 showed that an orifice without sharp corners greatly reduced the more intense electric field at the entry and exit corners of the orifice. Thus a rounded corner orifice substantially eliminates the non-uniform sensitivity artifact but does not eliminate the recirculating particle artifact.
Thr designs reviewed above all suffer from significant shortcomings. Front and rear sheath flow designs consume considerable quantities of special particle-free fluid which adds to the cost of operation as well as to the size and cost of the instrument system itself. It also adds to the cost of labor to fill the supply reservoirs, empty waste reservoirs, and dispose of large quantities of biohazardous waste fluid.
Designs using potential sensing electrodes within the orifice tend to be costly due to the difficulty of fabricating the orifice assembly itself. Also the design is not readily adaptable to the original Coulter volume sensing scheme having the orifice toatally immersed in electrolyte where the electrode leads must be well insulated from one another as well as the surrounding electrolyte and must have low interelectrode capacitance.
Designs using rounded corner orifice shapes are commonly used where only one population of particle sizes is to be encountered, such as erythrocytes or leukocytes. It can be fitted with rear sheath flow to eliminate recirculating particle artifacts to permit analyzing large and small particles simultaneously. However, this incurs the shortcomings due to use of sheath flows described above. Additionally, the rounded edge entrance tends to cause wedging of particles slightly larger than the orifice diameter which makes them more difficult to remove by wiping or reverse flushing than is true with sharp cornered orifices. Also partial clogs at the orifice inlet can cause significant changes in the orifice volume sensitivity.
My invention described herein provides the advantages and performance of orifices with front and rear sheath flow with none of their disadvantages.
Several objects and advantages of the particle volume sensing orifice arrangement of the present invention are:
(a) to provide a particle volume sensing orifice arrangement with substantially uniform sensitivity to particle volume regardless of the path of the particle passing through the orifice;
(b) to provide a particle volume sensing arrangement free of substantial response to particles recirculating downstream of the orifice near the orifice exit;
(c) to provide a particle volume sensing orifice arrangement which achieves the objects of (a) and (b) without use of front or rear sheath flows;
(d) to provide a particle volume sensing arrangement which achieves the objects of (c) which can easily replace existing orifices;
(e) to provide a particle volume sensing orifice arrangement with sharp entry corners which is more resistant to particle clogging and which is more readily cleared of clogging particles by wiping or reverse flushing than an orifice with rounded entry corners;
(f) to provide a particle volume sensing orifice arrangement which is not substantially affected as regards its particle volume sensitivity by the presence of a partially clogging particle at the orifice inlet;
(g) to provide a particle volume sensing orifice arrangement which provides the foregoing advantages with only a small increase in manufacturing cost;
(h) to provide a particle volume sensing orifice arrangement which provides the foregoing advantages and is useable in existing instrument designs with only negligible instrument design changes.
Further objects and advantages will become apparent from a consideration of the ensuing description and drawings.